Progress in molecular biology and translational science, volume 131

projection neurons have axons that cross the midline and ascend to multipleareas of the brain including the thalamus, periaqueductal gray matter, lateralparabrachial area of the pons, an

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Department of Pharmacology, The University of Iowa Roy J and Lucile A Carver College

of Medicine, Iowa City, Iowa, and Department of Anesthesiology, Washington University School of Medicine, St Louis, Missouri, USA

Durga P Mohapatra

Department of Pharmacology; Department of Anesthesia, The University of Iowa Roy J and Lucile A Carver College of Medicine, Iowa City, Iowa, and Department of Anesthesiology, Washington University School of Medicine, St Louis, Missouri, USA

Department of Pharmacology, The University of Iowa Roy J and Lucile A Carver College

of Medicine, Iowa City, Iowa, and Department of Anesthesiology, Washington University School of Medicine, St Louis, Missouri, USA

Justin Sirianni

Department of Anesthesiology, University of Arizona, Tucson, Arizona, USA

Andrew Michael Tan

Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven; Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare System, West Haven, and Hopkins School, New Haven, Connecticut, USA

When we were approached to assemble and edit this volume, we wereimmediately faced with a dilemma: there are many excellent pain textbooksthat already exist, why should we set out to create a new one? After sometime, and some perusing of the venerableTextbook of Pain, which has justreceived a fresh update, it occurred to us that there was indeed room for

a new textbook on pain While the existing texts are unquestionably lent, they largely fail to touch on exciting new areas of research emergingfrom young investigators in the field There are many young investigatorswho were on the frontlines of pain research in the laboratories of well-known figures in the field and now have their own independent laboratoriescontinuing their exciting lines of investigation Therefore, we decided to setout to create a textbook with a slightly different agenda Rather than focus-ing on specific topics, we decided to assemble a group of the leading younginvestigators in the field of pain research, give them some guidance on ouroverall goals, and set them loose to create the chapters they would like to seebased on their most exciting new areas of research

excel-The title of this book is: “Molecular and Cell Biology of Pain.” A bookwith such a title could have 100 chapters and take up an entire bookshelf Thisvolume is not meant to be comprehensive, not by any stretch of the imagi-nation, but it is meant to be exciting and new We hope that these are topicsthat are largely not covered in existing texts in the field but we also hope thatthese topics will have staying power in the field To that end, the group ofinvestigators assembled for this volume have already agreed, in principle,

to update this volume periodically as our research areas continue to progress

We hope that the evolution of this title over the coming years will give a sort

of history of the research endeavors engaged by the authors of these chapters

We are indebted to our mentors, who are many and whom we assumeknow who they are by this point We are also indebted to the institutions,University of Arizona and University of Texas at Dallas that have given usthe opportunity to do this work We would like to thank the production team

at Elsevier and especially Helene Kabes for her work and collaboration on thisproject Most importantly, we want to thank our colleagues who agreed totake on this project and without whom this volume most certainly wouldnot exist We value your academic collaboration, your scientific accomplish-ments at such an early career stage, and your continued friendship

xvii

Finally, it is our sincere hope that this book will find its way into lecturehalls throughout the world We have aimed the material at graduate andmedical students and we think it can give these students an interesting snap-shot of the forefront of the field If you are a student reading this foreword,

we hope you find something in this book that inspires discovery All of usstarted in your shoes aspiring to learn and make a contribution to mankindthrough scientific discovery We wish you the best, and we look forward toreading the volume that will be written by the coming generations

THEODOREJ PRICE

GREGORYDUSSOR

An Introduction to Pain Pathways

phys-Progress in Molecular Biology and Translational Science, Volume 131 # 2015 Elsevier Inc.

some neurotransmitters, are further discussed in the context of their relevance as tial drug targets for the better treatment of pain in patients with persistent pain Finally, this chapter introduces several important concepts in pain research that will be primary topics for chapters that come later in the book.

poten-1 AN INTRODUCTION TO PAIN AND PAIN PATHWAYSThe International Association for the Study of Pain (IASP) has definedpain as “an unpleasant sensory and emotional experience associated withactual or potential tissue damage, or described in terms of such damage.”1When asked to describe their pain, individuals variously described it in terms

of severity (mild, moderate, severe), duration (acute or chronic), and type(nociceptive, inflammatory, neuropathic).2

Nociceptive pain is the normal acute pain sensation produced by tion of nociceptors in skin, viscera, and other internal organs in the absence

activa-of sensitization.3–7It may occur as a result of mechanical, thermal, or ical noxious stimulation and is variously described as an aching or throbbingkind of pain.5,6,8,9Nociceptive pain comprises four main stages: transduc-tion (i.e., action at receptors in the periphery), transmission (i.e., actionpotentials along axons), perception (i.e., cortical processing of nociceptiveinput), and modulation (i.e., engagement of descending circuits).4,10–12Noxious stimuli are first detected by mechanical, thermal, and chemicalnociceptors found on specialized nerve endings present in skin (cutaneous),viscera, and other internal or external organs.8,9,13,14Nociceptive impulsesare transmitted from the periphery to the spinal cord via primary afferentnerve fibers which may be unmyelinated or myelinated.3,15–20The centralnervous system (CNS) components of this pathway constitute particularanatomical connections in the spinal cord, brain stem, thalamus, and cortex(the “pain pathway”), linking the sensory inflow generated in high thresholdprimary afferents with those parts of the CNS responsible for consciousawareness of painful sensations21 (Fig 1) Unmyelinated nerve fibers aresmall diameter C-fibers with diameters in the range 0.4–1.2μm.22,23

chem-Myelinated primary afferent nerve fibers are the Að-fibers (2–6μm ter), whereas the thinly myelinated nerve fibers are the Aβ-fibers (>10 μmdiameter).23,24 Primary afferent C-fibers and Að-fibers are responsible fortransmission of noxious stimuli whereas Aβ-fibers transmit innocuous,mechanical stimuli such as touch.21–24Put simply, nociceptors collect infor-mation from noxious stimuli which are transmitted by C-fibers and

diame-Að-fibers through the dorsal root ganglia to the superficial laminae I/II ofthe dorsal horn of the spinal cord.20,23 Að-fibers transmit impulses fromthe dorsal horn to deeper laminae (III–IV) of the spinal cord and onto highercenters in the brain via the spinothalamic tracts.20Dorsal horn neurons com-prise (i) projection neurons, (ii) local interneurons, and (iii) propriospinalneurons.20,25Although projection neurons are the primary means for trans-ferring sensory information from the spinal cord to the brain, they are only asmall fraction of the total number of cells in the dorsal horn.23,26 Many

Figure 1 Simplified schematic diagram of the pain pathway Pain begins with detection

of damage or potentially damaging stimuli by nociceptive neurons in the periphery that can transduce this signal into transmission toward the CNS The first synapse in this pathway is in the dorsal horn, where these projection neurons can send pain-related information onto multiple brain areas Pain perception occurs in the brain and can

be modulated by different centers in the brain The brain also sends modulatory inputs back down to the spinal cord to induce pain modulation.

projection neurons have axons that cross the midline and ascend to multipleareas of the brain including the thalamus, periaqueductal gray matter, lateralparabrachial area of the pons, and various parts of the medullary reticularformation.27These neurons are also involved in activation of endogenousdescending inhibitory pathways that modulate dorsal horn neurons.26Activity-dependent synaptic plasticity in the spinal cord that generatespostinjury pain hypersensitivity together with the cellular and molecularmechanisms responsible for this form of neuronal plasticity are termed

“central sensitization.”21 Neuroplastic changes relating to the function,chemical profile, or structure of the peripheral nervous system areencompassed by the term “peripheral sensitization” and encompass changes

in receptor, ion-channel, and neurotransmitter expression levels.28,29Central sensitization in the spinal cord includes sensitization and disinhibi-tion mechanisms, and supraspinally there are functional changes such asenlargement of receptive fields.30,31 In the CNS, there are also changes inthe dynamic interplay between neuronal structures and activated glialcells,30,32,33 a topic covered in depth in Chapter “Nonneuronal CentralMechanisms of Pain: Glia and Immune Response” by E AlfonsoRomero-Sandoval and Sarah Sweitzer

Following tissue injury and inflammation, vasoactive mediators such ashistamine, substance P (SP), serotonin (5-HT), nitric oxide (NO), prosta-glandins (PGs), and bradykinin are released which activate nociceptorsresulting in nociception.13This in turn can induce release of pronociceptiveneurotransmitters such as SP, calcitonin gene-related peptide (CGRP),dynorphin (Dyn), neurokinin A (NKA), glutamate, adenosine triphosphate(ATP), NO, PGs, and neurotrophins such as brain-derived neurotropic fac-tor (BDNF), from primary afferents either in the periphery or at the first syn-apse in the dorsal horn of the spinal cord.13,20,22,34,35 More recently, theimportant role of proinflammatory cytokines (e.g., tumor necrosis factor-alpha (TNF-α), interleukin-1β, interleukin-18, etc.) in peripheral and cen-tral sensitization mechanisms associated with persistent pain states has begun

to be appreciated.36

Many C-fibers express transient receptor potential vanilloid 1 (TRPV1)receptors and hence are sensitive to the vanilloid, capsaicin, which is a high-affinity ligand for TRPV1 receptors.37TRPV1-expressing C-fibers may befurther subdivided into two major classes:

(i) those that contain the neuropeptides, SP, and CGRP, express the affinity nerve growth factor (NGF) receptor, TrkA, and are develop-mentally dependent on NGF,34,38,39and

high-(ii) those that express isolectin B4, (IB-4), the P2X3purinergic receptor,40fluoride-resistant acid phosphatase, do not contain SP or CGRP,41andare dependent on glial cell line-derived neurotrophic factor (GDNF).34

1.1 Neuropathic pain

The IASP has defined neuropathic pain as “Pain initiated or caused by a mary lesion or dysfunction in the nervous system.”1,42Neuropathic pain isvariously described by patients as having one or more of the following qual-ities: burning, tingling, electric shock like, and stabbing or pins andneedles.5,42,43The appearance of abnormal sensory signs such as allodynia

pri-in response to pri-innocuous (nonnoxious) stimulation and/or hyperalgesia pri-inresponse to noxious stimulation is common.43 When neuropathic pain isevoked, it may be classified as having dysesthetic, hyperalgesic, or allodynicproperties depending upon the dynamic or static characteristics of thestimulus.44

In recent years, it has begun to be appreciated that the pathobiology ofvarious neuropathic pain subtypes may differ.45,46Hence, multiple researchgroups have focussed on developing and validating rodent models of each ofthese neuropathic pain conditions.47,48These more relevant rodent models

of neuropathic pain have considerable potential not only in terms ofunraveling the neurobiology of each of these neuropathic pain subtypesbut also in terms of identifying novel targets for discovery of new efficaciousand well-tolerated analgesics to improve relief of these persistent painconditions.43,49

Inflammatory and neuroimmune mechanisms contribute to bothperipheral and central sensitization that underpin the pathobiology of neu-ropathic pain.50 Following peripheral nerve injury, inflammatory cellsincluding mast cells, neutrophils, macrophages, and T-lymphocytes contrib-ute to peripheral sensitization and hyperexcitability of injured and adjacentnoninjured primary afferent nerve fibers.50In the CNS, activation of glialcells including microglia and astrocytes leads to the production and secretion

of various proinflammatory mediators that promote neuroimmune tion and can sensitize the central terminals of primary afferent andsecond-order neurons to increase the intensity and duration of pain.50–53However, rodent models of neuropathic pain allow behavioral painresponses such as mechanical allodynia in response to application of a non-noxious stimulus (light pressure) to the hindpaws and/or hyperalgesia inresponse to application of noxious stimuli (pressure, heat, cold) to the

activa-hindpaws, to be quantified.43,49 The three most commonly used rodentmodels of neuropathic pain involve induction of a unilateral chronic con-striction injury of the sciatic nerve,54 partial sciatic nerve ligation,55 andL5 spinal nerve ligation.56 Over the past two decades, these models havebeen used to identify numerous neuropathic pain “targets” encompassingvarious receptors, ion channels, and enzymes, as well as to assess novel paintherapeutics in development.43,49

More recently, rodent models of varicella zoster virus-induced pathic pain, antiretroviral drug-induced neuropathic pain, cancerchemotherapy-induced neuropathic pain, bone cancer pain, and multiplesclerosis-induced neuropathic pain have been developed.43,57,58It is hopedthat through use of these more sophisticated rodent pain models, it will bepossible to gain an enhanced understanding of the pathobiology of theseconditions Additionally, it may be possible to identify novel “druggable”targets for drug discovery aimed at producing novel analgesics withimproved efficacy and reduced adverse-event profiles for improved relief

neuro-of these chronic pain conditions in the clinical setting.49,59

Persistent ongoing pain secondary to nerve injury is underpinned byconsiderable complexity and plasticity at multiple levels of the neu-raxis.49,58,60–62Following peripheral nerve injury, ectopic firing of injuredand uninjured afferents induces neuroplastic changes and “centralsensitization” in the spinal cord and the brain, underpinned by both neuro-nal and nonneuronal mechanisms49(Fig 2) Intensive research over the pasttwo decades has revealed a large number of receptors, enzymes, and ionchannels as potential novel targets for drug discovery programs aimed at

Figure 2 Simplified diagram of mechanisms of neuropathic pain Peripheral nerve injury causes many changes in the function and phenotype of injured fibers, but an important property for neuropathic pain is the generation of ectopic activity (action potentials without any stimulus, signified by lightning) This ectopic activity drives spon- taneous pain and plasticity in the dorsal horn and brain that may underlie clinical fea- tures like allodynia.

producing new drugs for the relief of neuropathic pain.63–65Although eral molecules have entered preclinical and clinical development, very fewhave been approved by regulatory agencies for clinical use.66 Hence, thislarge unmet medical need is driving research in this field.

sev-1.2 Inflammatory Pain

Inflammatory pain is precipitated by an insult to the integrity of tissues at thecellular level One of the characteristic features of inflammatory states is thatnormally innocuous stimuli can produce pain.67 Inflammation is classicallyassociated with pain (dolor), heat (calor), redness (rubor), swelling (tumor),and loss-of-function (function laesa).67,68 Examples of inflammatory paininclude pain secondary to tissue injury and infection as well as rheumatoidarthritis.69,70Following tissue injury, nociceptors in the affected tissue becomesensitized due to the release of proinflammatory mediators from damaged cellsand blood vessels at the site of injury as well as from immune cells that invadethe injured site.71 This topic is covered in detail in Chapter “PeripheralScaffolding and Signaling Pathways in Inflammatory Pain by Jeske Nathan.”Inflammatory mediators including protons, 5HT, histamine, adenosine,bradykinin, prostaglandin E2 (PGE2), NO, IL-1, TNF-α, interleukin-6(IL-6), leukemia inhibitory factor, and NGF5,72,73contribute to nociceptorsensitization so that innocuous stimuli are detected as painful (allodynia) orthere is an exaggerated response to noxious stimuli (hyperalgesia).34,74Thecentral terminals of primary afferent nerve fibers (first-order neurons) arelocated in the superficial layers (laminae I/II) of the dorsal horn of the spinalcord.13Synaptic input from these terminals to second-order neurons in thespinal cord transfers information created by action potentials in primaryafferents secondary to peripheral noxious stimuli (depending on intensityand duration), to the thalamus, and then onto the cerebral cortex in thebrain.13 Synaptic function at the central terminals of first-order neurons isregulated by neurotransmitter release, primarily involving glutamate, andneuroactive peptides like substance P and CGRP.60–62,75–78

In inflammatory pain, peripheral inflammation induces a phenotypicswitch in primary sensory neurons to induce a change in their neurochem-ical character and properties.62This topic is covered in some depth in Chap-ters “Translation Control of Chronic Pain by Ohannes K Melemedjian andArkady Khoutorsky,” “Regulation of Gene Expression and Pain States byEpigenetic Mechanisms by S.M Ge´ranton and K.K Tochiki,” and

“Commonalities between Pain and Memory Mechanisms and their ing for Understanding Chronic Pain by Theodore J Price and Kufreobong EInyang.” In brief, this is underpinned by alterations in transcription and

Mean-translation of various receptors and ion channels to induce central tion by virtue of a change in the level of synaptic input produced by the sen-sitized afferent nerve fibers62 (Fig 3) Put simply, continuous inputs fromsensitized nociceptive afferents can activate or trigger central sensitizationthat is characterized by a reduced threshold of dorsal horn neurons to nox-ious stimulation16,79–84(Fig 3) There is expansion of the receptive fields ofdorsal horn neurons,85,86 and temporal summation of slow postsynapticpotentials resulting in a cumulative depolarization and a prolonged after dis-charge or “wind up” of dorsal horn neurons.15There is also increased excit-ability of the flexion reflex in response to peripheral stimulation.82,87 Theneural mechanisms underlying central sensitization involve excitatory aminoacids (EAAs, e.g., mainlyL-glutamate) acting at AMPA and NMDA recep-tors with the net result being persistent activation of the NMDA receptor84

sensitiza-to allow Ca2+entry into neurons and activation of numerous intracellularsignaling pathways.62

SP-expressing C-fibers and BDNF-expressing dorsal root ganglion(DRG) neurons both have a significant role in inflammation-induced cen-tral sensitization after exposure of their peripheral terminals to inflammatorymediators and NGF released from immune cells.62,88,89 Ectopic firing ofsensitized terminals increase Aβ-mediated synaptic input to superficial dorsalhorn neurons62,90and induction of cyclooxygenase-2 (COX-2) expressionlevels to drive production of PGE2.62,91,92 Retrograde transport of NGFfrom the peripheral terminals of C-fibers to the dorsal root ganglia inducesupregulation of TRPV1 expression and activation (phosphorylation) of p38MAPK.39,93 This in turn leads to upregulated synthesis and release of

Figure 3 Simplified diagram of mechanisms of inflammatory pain Activation of immune cells during inflammation leads to the release of inflammatory mediators that act on nociceptors Many of these inflammatory mediators from immune cells directly activate or modulate the activity of nociceptors This can drive spontaneous pain and plasticity in the dorsal horn and brain that underlies clinical features including allodynia and hyperalgesia.

proinflammatory cytokines among which IL-1β and TNF-α contribute tothe development of central sensitization by enhancing excitatory and reduc-ing inhibitory currents, and by activating induction of COX-2.62,91,94As aresult, GluR2-containing AMPA receptor activation allows entry of Ca2+into neurons,62,95 and this mechanism generates as much Ca2+ influx asoccurs with NMDA receptor activation during inflammatory pain.62,95

2 ION CHANNELS, RECEPTORS, AND OTHER “TARGETS”FOR PERSISTENT INFLAMMATORY OR

NEUROPATHIC PAIN

Intensive research over the past two decades has revealed a vast array

of ion channels, receptors, transporters, and enzymes that are potential

“druggable” targets for use in discovery programs aimed at developingthe next generation of analgesic drugs.66 Examples of “pain targets” forpotential modulation by novel analgesic agents include voltage-gatedsodium channels (Nav1.3, Nav1.7, and Nav1.8),12voltage-gated potassiumchannels (Kv1.4) is the sole Kv1 subunit expressed in smaller diameter sen-sory neurons96–98suggesting that homomeric Kv1.4 channels predominate

in Aδ and C-fibers arising from these cells.97

By contrast, larger diameterneurons associated with mechanoreception and proprioception expresshigh levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 subunits,suggesting that heteromers of these subunits predominate on large, myelin-ated afferent axons that extend from these cells.97,99–102 Additional “paintargets” include voltage-gated calcium channels (VGCC) (α2δ subunits;

Cav2.2, Cav3.1, Cav3.2, Cav3.3),103 acid-sensing ion channels(ASICs)104covered to some extent in Chapter “Meningeal Afferent Signal-ing and the Pathophysiology of Migraine by Carolina Burgos-Vega, JamieMoy and Greg Dussor,” NMDA receptors, TRPV1 receptors, covered inChapter “Sensory TRP Channels: The Key Transducers of Nociceptionand Pain by Aaron D Mickle, Andrew J Shepherd and Durga P.Mohapatra,” NKA, purinergic receptors, toll-like receptors (TLRs),protease-activated receptors (PAR) receptors, opioid receptors, the norepi-nephrine transporter, and cyclooxygenases (COX-1/2).105 Many of these

“pain targets” are described briefly in the following sections and in moredetail throughout this volume, as noted and summarized inFig 4

2.1 Ion channels

Ion channels play a key role in nociception and are involved in sensorytransduction (TRPV1), regulation of neuronal excitability (potassium

channels), action potential propagation (sodium channels, ATP-gated nels, ASICs), and presynaptic release of various neurotransmitters (calciumchannels).106

chan-2.2 Sodium channels

Voltage-gated sodium channels are considered a major target for the opment of novel therapies for improving pain management, as the ectopicfiring of primary afferents is associated with abnormal sodium channelregulation.107–109

devel-Sodium channels comprise an α-subunit containing a voltage-gatedsodium-selective aqueous pore and one or two smaller ancillaryβ-subunits.110

Sodium channel subtypes differ in their sensitivity to block by tetrodotoxin(TTX) with six isoforms being sensitive (TTXs: Nav1.1, Nav1.2, Nav1.3,

Na 1.4, Na1.6, Na 1.7) to block by nanomolar concentrations of TTX

Figure 4 Mechanisms of inflammation-induced pain in the periphery Tissue damage causes an immune response and the release of inflammatory mediators that act on nociceptors As described in the text, peripheral nociceptors are finely tuned to detect mediators released by immune cells via the expression of receptors that bind these ligands and the presence of ion channels that are altered by the signaling pathways downstream of activation of these receptors.

and three isoforms being resistant to micromolar concentrations of TTX(Nav1.5, Nav1.8, Nav1.9).111

Acute inflammatory and neuropathic pain can be attenuated or abolished

by local treatment with sodium channel blockers,112showing that peripheralnociceptive input is dependent on the presence of functional voltage-gatedsodium channels Four voltage-gated sodium channel subtypes (Nav1.3,

Nav1.7, Nav1.8, and Nav1.9) are of greatest interest in pain due to theirselective expression in peripheral nerves.107,113–128For first-order sensoryneurons, Nav1.8 is expressed by the cell body, peripheral terminals, and cen-tral terminals within the dorsal horn of the spinal cord.110Anatomical andelectrophysiological evidence indicates that expression of Nav1.9 is largelyrestricted to nociceptive Aδ- and C-fibers.110

Highlighting the importance of Nav1.7 in inflammatory pain (Fig 4),levels of expression of Nav1.7 are increased in sensory nerve terminals byinflammation12,128and following ablation of Nav1.7 in nociceptive neurons,inflammatory pain responses are greatly reduced.129Additionally, dominantgain-of-function mutations in SCN9A, the gene encoding Nav1.7 toincrease DRG neuron excitability are thought to be causal in two inheritedchronic pain disorders in humans, viz, erythromelalgia, characterized byburning pain and skin redness in the extremities, and paroxysmal extremepain disorder, characterized by skin flushing, rectal, periocular, and peri-mandibular pain evoked principally by mechanical stimuli.109,130Addition-ally, congenital indifference to pain due to rare recessive loss-of-functionmutations in SCN9A mean that although individuals so-affected are of nor-mal intelligence, they often fail to recognize and report pain in response toinjury or infection which can lead to early mortality.131

Conditional knockout of SCN9A in mice abolished mechanical pain,inflammatory pain, and reflex withdrawal responses to noxious heat recapit-ulating the pain-free phenotype in humans with SCN9A loss-of-functionmutations.128Interestingly, as conditional knockout of SCN9A in both sen-sory and sympathetic neurons in mice with spinal nerve transection, mark-edly reduced neuropathic pain behavior in these animals, neuropathic paintherefore appears to involve interaction between sensory and sympatheticneurons.128

In neuropathic pain, levels of expression of Nav1.3 are increased in aged peripheral nerves and this is highly correlated with the appearance of arapidly repriming sodium current in small DRG neurons consistent with thenotion that Nav1.3 channels make a key contribution to neuronal hyper-excitability in neuropathic pain.12,105,110,132–135

to initiation of the action potential to regulate neuronal excitability.103,150–152Multiple studies in rodent pain models have implicated theα2δ1subunit

of presynaptic calcium channels as having an important role in persistent painstates.153 This is emphasized by clinical studies showing that the anti-neuropathic drugs, gabapentin, and pregabalin that are ligands at the α2δ1

subunit, have efficacy for the relief of neuropathic pain.154–157The anisms of action of these drugs are still controversial, but their widespreaduse for neuropathic pain disorders is highlighted in Chapter “Chronic PainSyndromes, Mechanisms, and Current Treatments by Justin Sirianni,Mohab Ibrahim and Amol Patwardhan.”

mech-2.4 K+channels

Voltage-gated K+(Kv) channel subunits are expressed in DRG neurons andhave an important physiological role in the regulation of membrane poten-tials in excitable tissues including nociceptive neurons.101,102,158–160The Kvchannel subunit Kv1.4 is the sole Kv1α subunit expressed in smaller diam-eter neurons, suggesting that homomeric Kv1.4 channels predominate in Aδand C-fibers arising from these cells.97,99,100Additionally, these neurons arepresumably nociceptors, because they also express the TRPV1 capsaicinreceptor, CGRP, and/or Na+ channel SNS/PN3/Nav1.8.97,99,161–164However, larger diameter neurons associated with mechanoception andproprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 orother Kv1α subunits, suggesting that heteromers of these subunits predom-inate on large, myelinated afferent axons that extend from thesecells.97,99,100,164As the opening of K+ channels leads to hyperpolarization

of the cell membrane and so decreased nerve cell excitability, several Kvchannels are implicated as possible targets for novel pain therapeutics For

example, A-type potassium currents contribute significantly to neuronalexcitability and central sensitization in the dorsal horn of the spinal cord

in inflammatory pain.100,165–169

However, abnormal hyperexcitability of primary sensory neurons plays

an important role in neuropathic pain.164,166Kv channels regulate neuronalexcitability by affecting the resting membrane potential and influencing therepolarization and frequency of the action potential and may therefore play akey role in ectopic activity that develops in peripheral nerves driving neu-ropathic pain.163,164 Additionally, diabetes primarily reduces Kv channelactivity in medium and large DRG neurons.164 Increased BDNF activity

in these neurons likely contributes to the reduction in Kv channel functionthrough TrkB receptor stimulation in painful diabetic neuropathy.164

2.5 Receptors

In inflammatory pain, multiple receptor classes located on nociceptors aremodulated by vasoactive mediators released from damaged tissues andimmune cells that invade the inflamed tissues.170

2.6 Purinergic receptors

ATP activates P2X purinergic receptors, especially P2X1, P2X3, or P2X7

receptors, to produce pain.171–173Currently, a range of preclinical studiesare investigating a role for P2X receptors in pain, inflammation, osteoporo-sis, multiple sclerosis, spinal cord injury, and bladder dysfunction.173Some

of these have been progressed into clinical trials for rheumatoid arthritis,pain, and cough.173

P2X3 receptors, located exclusively on small diameter fibers, are implicated in inflammatory pain and P2X4receptors on microglia

nociceptive-in the dorsal horn of the spnociceptive-inal cord are implicated nociceptive-in the pathogenesis ofneuropathic pain.174 Antagonists at the P2X7 receptor also reduce painbehaviors in rodent models of inflammatory and neuropathic pain, againhighlighting that purinergic glial–neuronal interactions are important mod-ulators of noxious nociceptive neurotransmission.175Hence, P2X3, P2X4,and P2X7receptors are potential targets for novel therapeutics for the treat-ment of inflammatory and neuropathic pain conditions.176,177

2.7 Toll-like receptors

Proinflammatory central immune signaling contributes significantly to theinitiation and maintenance of heightened pain states because recent

discoveries have implicated the innate immune system, in particular, patternrecognition TLRs in triggering these proinflammatory central immune sig-naling events.178There is considerable interest in the targeting of TLRs onimmune cells for the prevention and treatment of cancer, infection, inflam-mation, and autoimmune diseases.179In neuropathic pain, “TLR4 receptors

on peripheral immune cells (e.g., monocytes/macrophages, dendritic cells,and immune-related cells such as keratinocytes)”170 as well as “activatedmicroglia in the CNS” appear to have a key role in the establishment of thiscondition.180–183 Acute TLR4 antagonism attenuates neuropathic painbehavior and potentiates opioid antinociception.184Hence, TLR4 appears

to be a possible target for therapeutic intervention for relief of neuropathicpain and for augmenting opioid analgesia As already noted in an earlier sec-tion of this literature review, dysregulation of chemokines (Section 1) andtheir receptors (Section 2.1;Fig 4), particularly fractalkine and its CX3CR1receptor, appear to play a key role in neuroimmune signaling that contributessignificantly to the pathobiology of neuropathic pain.185 This target is dis-cussed in more detail in Chapter “Role of Extracellular Damage-AssociatedMolecular Pattern Molecules (DAMPS) as Mediators of Persistent Pain byJungo Kato and Camilla I Svensson.”

2.8 PAR receptors

PARs are G-protein coupled receptors (GPCRs) that have a unique tion mechanism involving specific proteolytic cleavage of the amino-terminalsequence by serine proteases.186–188PAR1is expressed by primary afferentneurons and can modulate nociception.189 PAR2 is expressed by SP- andCGRP-containing primary afferents.189 Activation of PAR2 induces therelease of the pronociceptive neurotransmitters, SP, and CGRP from bothperipheral and central terminals of primary DRG neurons.189PAR4modu-lates nociceptive responses in normal and inflammatory conditions such that aPAR4agonist alleviated inflammatory pain in rats.159,188,190–196

activa-2.9 Glutamate receptors

Glutamate is the major EAA neurotransmitter in the central nervous systemand is found in at least 70% of sensory neurons in the DRGs.197It is releasedfrom the central terminals of primary afferents and has an important role innociceptive neurotransmission.197 Glutamate acts via two main receptorclasses, iGluRs (ionotropic), and mGluRs (metabotropic) with iGluRs fur-ther subdivided into AMPA, NMDA, and kainate receptors.72,198 AMPA

and NMDA receptors are directly coupled to cation-permeable ion channelswhereas metabotropic glutamate receptors (mGluRs) are coupled viaG-proteins to soluble second messengers.53,72,198–200Brief nociceptive stim-uli primarily activate AMPA receptors whereas stimuli of more prolongedduration activate NMDA receptors.35,201Astroglial cells remove excess glu-tamate from the extracellular space and express the glutamate reuptake trans-porters, GLAST/EEAT-1 (excitatory amino acid transporter) and glutamatetransporters EEAT-2/GLT-1.202–207

2.10 AMPA receptors

AMPA receptors have an important role in acute spinal processing of ceptive and nonnociceptive inputs.208 Activation of AMPA receptors byglutamate results in potent depolarization of dorsal horn neurons to removethe Mg2+block, and hence activate NMDA receptors resulting in calciuminflux and initiation of a cascade of downstream signaling events.209BecauseAMPA receptors also have roles in many other CNS functions,208they aregenerally regarded as being unsuitable targets for development of novel paintherapeutics.208

“central sensitization” in the spinal cord in persistent pain states.215Althoughmolecules targeting NMDA receptors have potential for the relief of persis-tent pains such as neuropathic pain, NMDA receptors are involved in nor-mal physiological functions and so the first generation of these agents werehampered by CNS side effects in the analgesic dose range.216

2.12 Metabotropic glutamate receptors

Group I mGluRs in laminae I/II of the spinal dorsal horn play an importantrole in the transduction of nociceptive input from C-fibers.217 There arethree mGluR classes containing eight cloned mGluRs and in vivo studiesshow that these are not involved in acute nociceptive signaling.218–220Group I mGluRs (mGlu1 and 5) are implicated in central sensitization

and persistent nociception.221By contrast, activation of group II mGluRs(mGlu2/3) alleviates neuropathic and inflammatory pain.221 This topic isdiscussed in more detail in Chapter “mGluRs Head to Toe in Pain byBenedict J Kolber.”

2.13 Opioid receptors

Opioid receptors are members of the superfamily of 7-transmembrane ning region GPCRs that are coupled to intracellular effectors viaG-proteins, mainly of the inhibitory type, i.e., Gi,o.25,222,223There are highdensities of opioid receptors in various brain regions, the dorsal horn of thespinal cord, on peripheral nerve terminals, in peripheral tissues including thegastrointestinal tract and on immune cells (Fig 2).198,224,225

span-In the 1990s, three opioid receptor types, viz,μ (MOP), δ (DOP), and κ(KOP) were cloned, and more recently multiple splice variants of thesereceptors, particularly the MOP receptor, have been identified.222,226–228The endogenous ligands for opioid receptors include the endomorphins(highly MOP selective), β-endorphin (equal MOP and DOP selectivity),met- and leu-enkephalin (more selective at DOP than MOP), and Dyn(KOP selective).229–231Opioid agonists activate opioid receptors to producepotent analgesia by activation of the descending inhibitory system to inhibitascending excitatory nociceptive transmission (Fig 2).224,232,233 Studiesusing MOP receptor knockout mice show that the antinociceptive andother effects of morphine and most other clinically available opioid analge-sics, are produced secondary to activation of the MOP recep-tor.222,223,234–239 Studies using rodents show that peripheral opioidactions are increased in inflammation, suggesting that peripherally selectiveopioid analgesics may have benefit as future analgesic agents that are devoid

of CNS side effects.25,222

2.14 TRPV receptors

TRPV1 receptor (Fig 4) is a ligand-gated nonselective cation channelexpressed on primary afferent sensory neurones that can be activated byexogenous agents (e.g., capsaicin), endogenous substances (e.g., bradykinin,ethanol, nicotine, anandamide, and insulin) as well as by heat (>43 °C) andlow pH.240–243Following activation of TRPV1, there is a rapid increase inintracellular Ca2+concentrations resulting in nociceptive signal transductionvia C-fibers to produce pain in humans and pain behaviors in animals.243,244TRPV1 knockout mice exhibit reduced thermal nociception and a loss ofinflammatory thermal hyperalgesia.242,245However, both NGF and GDNF

elicit thermal hyperalgesia during peripheral inflammation via an increase inTRPV1 expression.246Expression of these two growth factors follows dif-ferent time courses and they act on distinctive subpopulations of DRG neu-rons.246 Intradermal injection of capsaicin and NGF produce heathyperalgesia via activation of their respective receptors, viz, TRPV1 andTrkA on sensory nerve terminals Moreover, PI3K induces heathyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependentmanner, and the PI3K pathway also appears to play a role that is distinct fromERK (Fig 4) by regulating the early onset of inflammatory pain.39,93,247,248TRP channels are discussed in more detail in Chapter “Sensory TRP Chan-nels: The Key Transducers of Nociception and Pain by Aaron D Mickle,Andrew J Shepherd and Durga P Mohapatra.”

2.15 Prostaglandin (prostanoid) E2

Inflammatory pain hypersensitivity is regulated by prostaglandin receptors(EP1, EP2, EP3, EP4 receptors; Fig 4).249 At the site of inflammation,PGE2sensitizes peripheral nociceptors via activation of EP2 receptors thatare present on the peripheral terminals of high threshold sensory nerve fibers

by reducing the nerve firing threshold and increasing responsiveness, which

is the key phenomenon of peripheral sensitization.249,250

Following tissue injury, the synthesis of PGE2in the spinal cord91tributes to central sensitization251and increased excitability of spinal dorsalhorn neurons.249 NSAIDs inhibit prostaglandin synthesis through non-selective inhibition of constitutively expressed cyclooxygenase COX-1 aswell as the inducible isoform COX-2.252–254

con-2.16 Pronociceptive neurotransmitters

2.16.1 Nitric oxide

In the CNS, NO is synthesized primarily from the precursor,L-arginine, bythe enzyme nitric oxide synthase (NOS).255There are three NOS isoforms,viz, neuronal (nNOS), endothelial (eNOS), and inducible nitric oxidesynthase (iNOS), with nNOS having a role in the modulation of nociceptivetransmission in the spinal cord.255,256Following glial cell activation in theCNS, NO is produced by iNOS, to further sensitize nociceptive neuronesand contribute to the maintenance of central sensitization in persistent painconditions.256NO produced in excess by iNOS and nNOS is implicated ininflammatory and neuropathic pain, and so iNOS and nNOS inhibitors havebeen investigated as potential novel agents for alleviation of these chronicpain conditions.257

2.16.2 Nerve growth factor

NGF levels increase during inflammation258–260resulting in sensitization ofprimary afferents to noxious thermal, mechanical, and chemical stimuli viaupregulated synthesis of TRPV1 receptors as well as SP, CGRP, and bra-dykinin receptors261–265 (Fig 4) Anti-NGF therapeutics have advanced

to clinical trials in humans and have shown broad efficacy but also severe,albeit rare, side effects NGF signaling is discussed in more detail in Chapters

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Peripheral Scaffolding and

Progress in Molecular Biology and Translational Science, Volume 131 # 2015 Elsevier Inc.

peripheral afferent neuronal tissues specifically are identified and discussed Together, the mediators, pathways, and scaffolding mechanisms involved in inflammatory hyperalgesia provide a unique knowledge point from which new therapeutic targets can be understood.

1 INTRODUCTION

Human physiology integrates multiple redundant homeopathic naling mechanisms that maintain tissue structure and function One suchmechanism, inflammation, occurs in response to tissue damage and is stim-ulated by the generation of an immune response Following injury, neutro-phils, leukocytes, and other immune cells release and stimulate the release ofsignaling factors from adjacent tissues These inflammatory signaling factorsactivate receptors located on terminal endings of primary afferent fibers,which in turn activate intracellular mechanisms that sensitize additionalreceptors and ion channels In the end, a state of inflammatory hyperalgesia,

sig-or increased sensitivity to pain following an inflammatsig-ory response, motes homeostatic behavior through stimulus avoidance of the inflamedand/or the injured tissue

pro-The activation of proinflammatory receptor systems creates hyperalgesicconditions to reduce the likelihood of further injury to the damaged periph-eral tissue Most mediators released into the extracellular space surroundinginjured tissue activate receptor systems expressed by primary afferent neu-rons that sensitize those neurons to physical and chemical stimuli In mostcases, this occurs through posttranslational modifications to ion channels,reducing either the ligand dependency or the voltage dependency requiredfor activation and eventual depolarization of the neuron The phenotype ofinflammatory hyperalgesia is marked by increased sensitivity to a normallypainful stimulus and is typically supported by reversible changes to intra-neuronal biochemistry In this chapter, these molecules, their receptors, sig-naling pathways, and scaffolding complexes will be identified andcharacterized for their roles in inflammatory hyperalgesia

2 INFLAMMATORY MEDIATORS

Injury to peripheral tissue elicits changes to the extracellular istry surrounding the tissue and associated primary afferents that innervatethe tissue Damaged epithelial cells, in concert with responding immune

biochem-cells, release small peptides and molecules that target receptors expressedthroughout the periphery These receptors control peripheral vasodilation,microglial response, epithelial cell repair, and nociceptor sensitivity Asinflammatory mediators such as bradykinin and endothelin (ET) increasevasodilation, plasma extravasation brings additional immune cells andinflammatory mediators into the area of the tissue injury, creating a feed-forward inflammatory response The activation of microglia by inflamma-tory mediators can result in the release of molecules such as prostaglandins(PGs) and adenosine triphosphate (ATP), both of which serve to activatespecific receptors expressed on primary afferent neurons The activation

of these receptor systems, along with others that are activated by numerousother inflammatory mediators, stimulates intracellular signaling pathwaysthat increase nociceptor sensitivity, resulting in hypersensitization In such

an environment, the nociceptive terminal ending is highly sensitive toreduced thresholds for somatosensory and chemical activation, causingincreased pain InTable 1, a list of inflammatory mediators and their targetreceptor systems is outlined

The process of peripheral inflammation is a feed-forward mechanism inits execution and end point The local invasion of immune cells in response

to tissue injury results in the release of molecules such as serotonin and serineproteases These initial immune factors act upon their respective receptorsexpressed on terminal endings of nociceptors to sensitize neuronal depolar-ization and the subsequent release of substance P, CGRP, PGs, and gluta-mate These nociceptor-originated mediators then act to perpetuate thesensitization of terminal nociceptors through receptor activation and also

by stimulating vasodilation, thereby increasing the delivery of circulatingimmune cells and inflammatory peptides to the area of injury As depicted

inTable 1, most inflammatory mediators target specific isoforms of receptorsexpressed by nociceptors and other cell types Receptor activation is whatdrives inflammatory sensitization of peripheral tissue, as ligands make con-tact with specific receptors that dictate downstream signaling systems.Inflammatory sensitization of primary afferent nociceptors can onlyoccur if the receptor system that the inflammatory mediator is directedtoward is mated to a stimulatory signaling cascade Indeed, several specificisoforms of the serotonin receptor have been identified as regulating the sen-sitizing behavior of serotonin on nociceptor functions Depending on theisoform activated, either Gαq- or Gαs-driven downstream signaling wouldoccur, resulting in the sensitization of differentially unique targets within thenociceptor itself Other inflammatory mediator receptor isoforms do not

Bradykinin Immune

cells, plasma

Blood vessels, nociceptors

Bradykinin type 1 and 2 receptors (B1R, B2R)

LOX

Thermal, mechanical

nociceptors

Nociceptors, immune cells, microglia